To further explore the function of CBD103
in an experimental genetic system, we generated transgenic mice in which a cDNA encoding either the KB
or the ky
allele was driven by a strong and widely expressed promoter (22
). We chose a genetic background that normally has Agouti-banded hairs and observed that two transgenic founders generated with the CBD103
ΔG23 cDNA (the KB
allele) displayed a predominantly black coat with small patches of banded hair (). Unexpectedly, the normal CBD103
cDNA (the ky
allele) also produced transgenic mice with a black coat in 20 out of 21 founders (). Furthermore, we observed that transgenic animals were smaller than their nontransgenic littermates. By 2 weeks of age, female transgenic animals were easily recognized by their dark coat and small size; in adult mice, reduced body weight persists in both males and females (fig. S7
Pigmentary effects of CBD103 in transgenic mice. Photographs of transgenic (Tg) and non-transgenic littermates, representative of 2/2 independent founders for Tg.CBD103ΔG23 and 20/21 independent founders for Tg.CBD103.
These considerations suggest three possible mechanisms by which CBD103 might act to modulate melanocortin signaling: (i) by binding to and activating the Mc1r, (ii) by binding to the Mc1r and preventing its inhibition by Agouti protein, or (iii) by binding to Agouti protein leading to its sequestration and/or degradation. To distinguish among these ideas, we generated synthetic forms of CBD103 and tested their ability to interact with the Mc1r and agouti signaling protein–YY (ASIP-YY), a synthetic version of the C terminus of Agouti protein that behaves as a competitive antagonist of α-MSH at the Mc1r and Mc4r (23
The CBD103 ky
allele predicts a mature peptide (after signal sequence cleavage) of 45 amino acids that contains six cysteine residues and begins with the glycine that is deleted in the KB
allele (). The predicted signal sequence cleavage site is supported by biochemical studies of the orthologous human protein [known as DEFB103 or human β-defensin 3 (HBD3)] purified from human tissues (24
). We synthesized the 45-residue ky
form and 44-residue KB
form of the dog protein (hence referred to as CBD103 and CBD103DG23, respectively), allowed oxidative refolding, and used mass spectrometry and high-performance liquid chromatography to confirm recovery of a single congener with three intrachain disulfide bonds.
]-α–MSH (NDP-MSH), a potent derivative of α-MSH, stimulates robust accumulation of cyclic adenosine monophosphate (cAMP) in melanocytes (25
). However, neither CBD103 nor CBD103ΔG23 has any effect on cAMP accumulation (), indicating that CBD103 is not a conventional Mc1r agonist. To test for receptor binding, we transiently transfected human embryonic kidney (HEK) 293 cells with Mc1r expression constructs (22
) and used europium-labeled NDP-MSH (Eu-NDP-MSH) as a fluorescent tracer. We used saturation binding assays to first calculate the affinity of Eu-NDP-MSH for the dog Mc1r () and then carried out displacement assays with progressively increasing concentrations of CBD103, CBD103ΔG23, or ASIP-YY. All three peptides exhibited qualitatively similar profiles characteristic for competitive binding to a single high-affinity site ( and ). Quantitatively, inhibition constant (Ki
) values estimated from the data depicted in were 150.6 nM for CBD103 and 34.2 nM for CBD103ΔG23. These estimates varied according to experimental conditions, but CBD103ΔG23 consistently exhibited higher affinity for the dog Mc1r than did CBD103 (mean of fivefold across four paired experiments; ). In these same experiments, ASIP-YY exhibited Ki
values of 0.51 to 0.95 nM for the dog Mc1r, in the same range as reported previously for ASIP-YY at the human Mc1r (23
). Using quantitative RT-PCR, we found that the levels of CBD103
mRNA in total skin were ~300-fold greater than that of Agouti
mRNA; to the extent that this difference reflects protein abundance, the levels of CBD103ΔG23 in dog skin are likely to be much greater than those of Agouti protein, which is consistent with a model in which CBD103ΔG23 competitively inhibits the ability of Agouti protein to antagonize Mc1r signaling.
Fig 5 Pharmacology of β-defensin action on melanocortin receptors. (A) Ability of NDP-MSH or CBD103 to stimulate cAMP accumulation in cultured melanocytes. (B) Saturation binding of Eu-NDP-MSH to HEK293 cells transiently transfected with the dog Mc1r. (more ...)
Table 1 Affinity constants for melanocortin receptor ligands. In the column for Eu-NDP-MSH, saturation binding assays as depicted in were used to derive dissociation constant (Kd) values (in nM) by fitting the data to a hyperbolic dose-response curve (more ...)
Finally, we investigated a potential interaction between Agouti protein and CBD103 by preincubating ASIP-YY with either CBD103 or CBD103ΔG23 before the binding assay, and we found no evidence for a functional interaction (fig. S8
). We also examined the effects of mixing the two peptides on two-dimensional nuclear magnetic resonance spectra and found no evidence for a structural interaction. Taken together, these observations indicate that CBD103 is a high-affinity ligand for the Mc1r and that the ΔG23 mutation may cause a black coat color in dogs via two different mechanisms: increased affinity for the Mc1r and increased availability of the mature protein in vivo.